1. Field of the Invention
The present invention relates to the field of molecular and cellular biology. More specifically, the present invention relates to a mutant blue fluorescent protein (BFP) capable of existing in an anaerobic or aerobic system and methods of using the same for fluorescence resonance energy transfer (FRET) and a blue fluorescent fish.
2. Description of Related Art
Fluorescent proteins, such as green fluorescent proteins (GFPs) from Aequorea victoria or GFP-like fluorescent proteins, have become an invaluable tool in cell biology. Over the last several years, GFP variants with altered fluorescence spectra, improved folding properties, increased brightness and altered pH-sensitivity have been increased (Tsien, 1998; Chudakov et al., 2005), and these GFP variants are widely used in the fields of biochemistry, molecular and cell biology, medical diagnostics and drug screening methodologies.
When GFP family proteins are used as reporter molecules, newly-synthesized GFP family polypeptides need to mature properly before emitting fluorescence. The maturation involves two steps: firstly, the protein folding into a nearly native conformation, and then cyclization of an internal tripeptide followed by oxidation. Therefore, the intrinsic brightness of the GFP family proteins in mammalian cells are determined by their expressions, efficient foldings and maturations at 37° C. An additional factor affecting the brightness of GFP family proteins in living organisms is that they strictly require oxygen as a cofactor for fluorescence formation. In fact, all members of the GFP family lose their luminance under rigorously anoxic conditions (<0.75 μM O2.) (Hansen et al., 2001). However, as described in the previous reports of the present inventors (Chang et al., 2004 (vol. 322); Chang et al., 2004 (vol. 319)), a blue fluorescent protein, BfgV found from Vibrio vulnificus, fluoresces through augmenting the intrinsic fluorescence of NADPH bound to it. Since NADPH is a common cofactor in most living organisms whether they are aerobic or anaerobic, BfgV and its improved variant, D7, can theoretically fluoresce in both aerobic living cells and anaerobic living cells (e.g. cancer cells). Consequently, BfgV variants with improved folding properties and increased brightness would be valuable in multicolor fluorescence experiments that allow in vivo labeling and detection in both the presence and absence of oxygen.
One technique for monitoring protein-protein interactions in both in vitro and in vivo assays is based on fluorescence resonance energy transfer (FRET). In this process, energy will transfer from one fluorophore (donor) to another (acceptor) when the donor emission spectrum significantly overlaps the acceptor absorption spectrum by a considerable percentage (30%) and these two fluorophores are closely approximated (within 10 nm). Fluorescent proteins with different emission wavelengths across the visible spectrum provide a variety of suitable donor-acceptor pairs for FRET.
Various methods of FRET measurements have been used to visualize protein-protein interactions. Recently, 3-FRET method that is capable of measuring FRET signals within a system of three donor-acceptor pairs, such as BFP coupled with GFP, cyan fluorescent protein (CFP) coupled with yellow fluorescent protein (YFP) and GFP coupled with red fluorescent protein (RFP), and multiple-FRET imaging by using two independently excitable FRET pairs have been reported. A bright and reasonably photostable fluorescent protein with fluorescence at ˜450 nm would be valuable in multicolor fluorescence experiments. Among the fluorescent proteins reported to date, a blue fluorescent protein (BFP) with excitation and emission maxima at 380 and 446 nm, respectively, which was developed from wild-type GFP by substitution of tyrosine 66, is particularly interesting because it is expected to be suitably paired with the most frequently used fluorescent proteins, EGFPs (enhanced GFPs), for multicolor imaging. However, BFP is dimly fluorescent in vitro and in vivo. Although a few enhanced BFPs (EBFPs) have been developed by introducing several mutations into BFPs, EBFPs are rarely used so far because of still having a undesirably low fluorescence quantum yield (QY), thereby being weakly fluorescent, and remaining relatively sensitive to photobleaching (Kremers et al., 2007). Therefore, EBFPs with further improvements in both brightness (i.e. with reasonably high QY) and photostability would be desirable. Recently, an ultramarine fluorescent protein, Sirius, with increased photostability and pH insensitivity has been reported. Since Sirius has an emission peak at 424 nm, it is spectrally compatible for 2-color imaging with EGFP (Tomosugi et al., 2009).
In addition, one major drawback shared by most newly discovered wild-type fluorescent proteins is that they are dimeric. Generally, the proteins exist as homodimers. However, when more than one form of a given fluorescent protein is expressed in a single cell or is mixed in vitro, heterdimers can form if the dimerization interfaces for the different fluorescent proteins are complementary. Heterodimerization is undesirable when fluorescent proteins are used to be expressed as a fusion to another protein of interest or when they are used in FRET. Many of the wild-type fluorescent proteins, however, can be engineered into monomers or tandem dimmers, which can then undergo further optimization.
To date, there have been no reports of BFP mutants still having high fluorescence quantum yield (QY), enhanced fluorescence and slow photobleaching at not only high temperature condition (e.g. 37° C.) but also at an anaerobic environment. Such mutants would provide obvious and significant advantages for use as cell markers or protein expression indicators in prokaryotic and, especially, eukaryotic systems where the stand physiological temperature is 37° C. and some of which are anaerobic (e.g. cancer cells), and for applying for FRET well.